Cyc1, the structural gene for yeast iso-1-cytochrome c, has been cloned and sequenced. Isolated cyc1 DNA will serve as molecular hybridization probe to isolate and clone the structural gene for yeast iso-2-cytochrome c (cyc7). By DNA sequencing of wild-type and iso-2-up mutant cyc7 genes, we will study the molecular basis for the large differences in expression between the cyc1 and cyc7 isoloci. To map the DNA regions essential for gene function, yeast transformation and complementation tests will be made with cycl and cyc7-bearing DNA fragments terminally deleted to varying extents. Localized in vitro mutagenesis experiments will be used to identify the specific functions carried out by noncoding regions of the transcription unit. Each mutant clone will be introduced (without selection for cyc gene function) into cyc7 yeast recipient cells and the blocked step in gene expression identified, then correlated with the DNA sequence alteration in the mutant. The cytochrome c genes from other eukaryotes (c.f. Neurospora and Drosophila) will be cloned, using the cyc1 DNA as probe. Comparative DNA sequencing will be used as one approach to determine whether similar mechanisms operate in cytochrome c gene expression in yeast and in other eukaryotes. By transformation of cyc yeast recipients with non-yeast cytochrome c genes, experiments can be done to directly compare yeast gene expression mechanisms with those of other eukaryotes. Such experiments would also make it possible to study, in a totally in vivo situation (for example, with a Drosophila cytochrome c molecule inside a respiring yeast mitochondrion) the effect of evolutionary amino acid substitution upon cytochrome c function in electron transport.